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Direct evidence for a role for Ca2+ in amine storage granule secretion by human platelets
0049-3848/80/220437-10$02.00/O THROMBOSIS RESEARCH 20; 437-446, 1980 Printed in the USA. All rights reserved.Copyright (c) 1980 Pergamon Press Ltd
DIRECT EVIDENCE FOR A ROLE FOR CA2+ IN AMINE STORAGE GRANULE SECRETION BY HUMAN PLATELETS
D.E. KNIGHT and M.C. SCRUTION Departments of Physiology and Biochemistry King's College Strand London WC2R 2LS, U.K.
(Received 22.8.1980; in revised form 29.10.1980. Accepted by Editor A.L. Bloom) ABSTRACT Exposure of washed human platelets to a high voltage electric field renders these cells permeable to small molecules such as Ca EGTA. Such accessed platelets secrete 5-hydroxytryptamine (SHT) when challenged with lo-5M Ca2+ but do not release lactate dehydrogenase under these conditions. The secretion of 5HT induced by Ca2+ requires MgATP but is independent of synthesis of prostaglandinendoperoxides. Half maximal secretion of 5HT is observed in the presence of 1.9)rM Ca2+. These data suggest a direct involvement of Ca2+ in platelet amine granule secretion.
INTRODUCTION
Secretion of adenine nucleotides and 5-hydroxytryptamine (5HT) from the amine storage granules of human blood platelets can be induced by a strong agonist such as thrcmbin or by the divalent cation ionophore A-23187 in the absence of aggregation and in media devoid of Ca2+ (l-3). However, when weak agonists such as ADP or adrenaline are employed observation of amine storage granule secretion is dependent on a prior aggregatory response and on the presence of Ca2+ in the medium at concentrations approaching the mM range (4,5). It is therefore inferred fran these and other observations that Ca2+ is directly involved in the pranotion of platelet secretion as is the case for other cells (cf. 6). Direct experimental evidence for such a role is however lacking for the platelet, since studies of the secretory event are limited by the technical difficulties involved in by-passing the transport systems of the plasma membrane while still retaining a preparation which possesses reasonable physiological integrity. We have therefore
Key Words: Platelets, Secretion, Calcium 437
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modified electric field technique which has been employed in studies of the role of Ca2+ in adrenalmedullary secretion (7) for use with the blood platelets, and have been able to obtain both direct evidence for the role of Ca2+ in secretion of 5HT fran human platelets and also an estimate of the range of intracellular Ca2+ concentration over which secretion is induced.
METHODS
Blood obtained fran healthy drug free volunteers was anticoagulated by addition of 1/6th volume of acid citrate-dextrose (8) and platelet-rich plasma prepared by centrifugation for 20 mins at 310 x g and then for 5 mins at 340 x g. After incubation for 30 mins at 37oC with 0.5 uCi [14C] - 5BT (creatinine sulphate) per 20 ml. of platelet-rich plasma platelets were collected by centrifugation for 25 mins at 415 x g and were resuspended in 0.28 M sucrose containing 20 mM K+PIPES, 20 mM K+ glutamate, 5 mM ATP, 7 mM MgCl2 and 5 mM glucose at pH 6.6 (buffer A). In a few experiments the resuspension medium contained 0.16 M K+ glutamate, 20 mM K+ PIPES, 5 mM ATP, 7 mM MgC12 and 5 mM glucose at pH 6.6 (buffer B). After addition of 10 mM EGTA the platelet plasma membrane was rendered leaky by exposure of these cells at 2OoC to an electric field generated by discharge of a 4.5 uF capacitor (previously charged to 1.5 KV) through 1.25 ml of platelet suspension (ca. 108 cells per ml.) contained between two parallel electrodes with a 1 mm gap. These conditions generate an electric field of 15 KV cm-l which decays with a calculated time constant of approximately 4 useconds (buffer B) or 30 useconds (buffer A). In theory exposure of the platelets to such a field should place a maximal potential difference of 2.25 volts across the plasma membrane at two sites, one at each end of the cell (9,lO). Dielectric breakdown, and hence localised membrane disruption, occurs at these sites as a result of this potential difference. The potential difference becomes established within the usecond exposure to the electric field since the time taken to charge up the membrane is calculated to be in the nanosecond range (9). Exposure to further discharges will be likely to produce additional points of membrane breakdown since the orientation of the cell will change with respect to the electric field. For adrenal medullary cells the diameter of the "holes" produced by similar membrane potentials has been estimated as being between 2 and 4 nm (9). The increase in temperature resulting frcm exposure of the platelet suspension to ten 15 KV discharges did not exceed 5oC. The extent of 5BT release was estimated from the [14C] content of the supernatant fraction after removal of the platelets by centrifugation at 8,000 x g. for 2 min. The [14C]content of the supernatant fraction remains constant after ccmpletion of release indicating that re-uptake of 5HT does not occur in platelets which have been exposed to the electric field. The Ca2+ concentration in the Ca EGTA buffers was estimated as described previously (11).
RESULTS
Two experimental approaches demonstrate that exposure of human platelets to an electric field as described above leads to localised, irreversible
439
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FIG. 1 Efflux and influx in leaky platelets A. Rb efflux from platelets rendered leaky by a single exposure to an electric field of 4 us duration and intensity 2.5 KVcm-1 (0 1, and 7.5 KVcm-1 (+ 1. Cells not exposed to the field (0 ). The suspending medium was plasma and the temperature 23OC. 45Ca EGTA influx into cells after 10 exposures to an electric B. field of 15 KVQU-1 ( V = 4 ps) ( ?? ). Cells not exposed to electric fields (0 ). Medium: Buffer B containing 10 mM EGTA and trace amaunts of 45Ca and 3H2O. Calculated free calcium a lo-gM. After exposure to the electric field, aliquots (0.2 ml.) of the platelet suspension were layered onto 0.2 ml of silicone oil (Versilube F50) and the cells separated by centrifugation at 8,000 x g for 2 mins.
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10 x 10 KV CM-’
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+-+-
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10S5M
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Ca
10-QM Ca +_
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(10 KVCti’)
FIG. 2 Ca2+ dependence of 5BT release The ordinates are expressed as % of the total 5HT in the suspension. Medium: Buffer B containing 10 mM CaEGTA to give calculated free Ca2+ of 10m5M ( 0 , 0 ) or lo-9M (a , 0 1. Open symbols: platelets not exposed to electric fields. A. 5HT in the supernatant fraction at different times after subjecting platelets to 10 exposures of 10 ICVC~-1 ( q = 4 us.) B. 5HT release in the supernatant fraction 5 minutes after subjecting platelets to the indicated number of discharges of 10 KVcm-1 (q = 4 us).
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First, platelets pre-loaded with 86Rb by incubation at membrane breakdown. 23oC for 30 mins rapidly release this radiolabel after a single exposure to a 7.5 KV cm-1 field (Fig. IA). Prolonged incubation following this exposure does not lead to re-uptake of 96Rb indicating that if membrane resealing has occurred, this process does not restore the ability to perform monoExposure to a lower field valent cation uptake using the Na+/K+ ATPase. (2.5 KVcm-1) causes a slower rate of S6Rb efflux from the platelets (Fig. 1A) suggesting a relationship between the strength of the applied field and the Second, platelets which do not normally take extent of membrane breakdown. up 45Ca in the resting state even in the absence of EGTA can be induced to take up 45Ca-EGTA rapidly in a system containing approximately 10sgM Ca2+ (free) on exposure to 10 discharges of a 15 ~vcm-1 field. In a system preequilibrated with 3H2O canparison of the 45Ca and 3H spaces before and after exposure to the field indicates that in excess of 65% of the intracellular environment of the platelets is made accessible to Ca-EGTA by this treatment (Fig. 1B). A slower rate, and possibly a decreased extent, of accessibility to 45Ca-EGTA is observed if the number of discharges or the intensity of the field is decreased. When platelets, previously loaded with [14C] - 5HT as described in "Methods", are rendered accessible to a Ca-EGTA buffer (10 mM EGTA) containing 70% of the [14C] 5HT is released to the 10m5M Ca2+ (free) approximately medium. In contrast, less than 5% release of [14C] - 5HT is observed when the Ca-EGTA buffer employed contains lo-9M Ca2+ (free) (Fig. 2A). This Ca2+-dependent release of [14C] - 5HT is rapid (Fig. 2A) and is not dependent on the discharge regime employed provided that the number of discharges are sufficient to ensure maximal accessibility to the Ca-EGTA buffer (Fig. 2B). The dependence of the release of [l+]-5HT on Ca2+ is confirmed by measurement of a dose/response curve with respect to Ca 2+ (free) concentration over the range 10sg to lo-5M Ca2+, (free) using the 10 mM Ca-EGTA buffer system. Fran the log dose/response relationship (Fig. 3) observed in several such experiments the Ca2+ (free) concentration required to induce 50% maximal release of [14C] - 5HT is obtained as 1.9 + 0.9 uM (S.E.M.) (n = 9). Fig. 3 also demonstrates that preincubation of the platelets with 10 pM indomethacin does not significantly affect the extent of Ca2+ -dependent 5HT release or the PreCa2' (free) concentration at which 50% maximal release is observed. incubation with indomethacin does however depress the extent of [ldc] - SHT release observed in the presence of lo-9M Ca2+ (free) and produces an approximately canparable depression of the extent of [14~] - 5HT release across the entire range of Ca2+ (free)concentration examined. Hence indomethacin is effective in this system. This observation therefore validates the conclusion that Ca2+ dependent 5HT release is unaffected by blockade of cyclooxygenase. TWO observations suggest that Ca 2+-dependent release of [14C] - 5HT fran human platelets is an exocytotic event. First, release of [14c] - 5HT induced by Ca2+ is not accanpanied by release of the cytosolic marker enzyme, lactate dehydrogenase (LDH) (Fig. 4). Second, observation of Ca2+dependent release of [14C] - ~HT is dependent on the availability of MgATP. The requirement for MgATP can be demonstrated by exposing platelets to the electric field in a buffer system containing 10~9M Ca2+ (free) and either no MgATP or 5 mM MgATP. After 5 minutes incubation in these buffers the accessed cells are then challenged by increasing the Ca2+ (free) concentration to 10s5M. Fig. 5A indicates that platelets exposed to the electric field in a buffer containing no MgATP rapidly lose their ability to release
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FIG. 3 Ca*+
activation curve for 5HT release
A suspension of platelets in medium A containing 0.3 mM EGTA was subjected to 5 discharges of 20 KVcm-1 ( q = 30 us) and aliquots then challenged with a range of 10 mM CaEGTA buffers. The 5HT in the supernatant fraction 5 minutes later was determined and is expressed on the ordinate as % of that in the suspension. Half the platelet suspension was preincubated with 10 PM indanethacin for 5 minutes before being exposed to the electric field ( 0 1. Platelets not incubated with indcmethacin ( 0 ).
- 5HT when the (free) Ca*+ concentration is subsequently increased to r% 10-5M. The extent to which responsiveness is lost is related to the degree of exposure to the electric field and hence possibly to the extent to which endcgeneous MgATP is lost. However loss of responsiveness is not observed if 5 mM MgATP is present in the suspending medium (Fig. 5A)_and after incubation in a medium lacking MgATP responsiveness to a Ca‘+ challenge may be restored by addition of MgATP. Fig. 5B shows a dose/response curve for such a restoration of responsiveness by MgATP for platelets exposed to 5 discharges of a 20 KVC~-~ field in a buffer lacking MgATP. Half maximal restoration of responsiveness is observed in the presence of 0.9 m!!MgATP.
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30
80
14C-5H T :/: 1:
60
40
50
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. /
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. 20
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-4
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FIG. 4 Ca2+ activation curve for 5HT ( 0
) and LDH ( 0 ) release.
A suspension of platelets in medium A containing 0.3 mM EGTA was subjected to a single discharge of 20 KVcm-1 ( ‘f = 30~s) and aliquots subsequently challenged with a range of 10 mM CaEGTA buffers. 5HT and LDH in the supernatant fraction were determined 5 minutes later and are expressed as percentages of the totals in the suspension.
DISCUSSION The studies described here demonstrate that the electric field technique which has previously been employed to probe secretory mechanisms in the adrenal medullary cells can also successfully be employed with only minor modification to examine this process in human blood platelets which are among the smallest of mammalian cells. Although the applied field is necessarily considerably more intense than that employed for adrenal medullary cells (7) the data obtained indicate clearly that functional integrity is preserved in the platelet at least in respect to the secretory mechanism. Minimal release of 5HT.results from exposure to the electric field in a buffer containing 10WgM Ca2+ indicating that as predicted (9,12) no significant breakdown of the membranes of the amine storage granules results fran exposure to the electric field. Furthermore the maximal extent of 5HT secretion obtained when accessed cells are challenged with 10m5M Ca2+ (free) is similar to that observed on stimulation of intact platelets with a saturating concentration of collagen, adrenaline or thranbin (13). These data also demonstrate that little, if any, extra-
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Mg-ATP (mM) FIG. 5 MgATP requirement for 5ET release A. Platelets were suspended in (i) Medium A containing 0.4 mM EGTA (+ 1, and in (ii) Medium A containing 0.4 W-lEGTA but without the The two suspensions were subjected to the 5 mM MgATP (0 ). indicated number of discharges at 20 Kvcm-1 ( q = 30 us) and were challenged 5 mins. later with 10 mM CaEGTA buffers corresponding to 10egM Ca2+. After a further 5 minutes the extracellular 5ET was determined, the difference in the levels for the two EGTA challenges representing the Ca2+ dependent release. Cells in Medium A containing 0.4 mM EGTA but no MgATP were -1 (q* 30 us) and one minute subjected to 5 exposures of 20 ICVcm later aliquots were incubated with various concentrations of MgATP. After a further 10 minutes the cells were challenged with 10 mM CaEGTA corresponding to 10-g and 10a5M Ca2+, and the Ca2+ dependent release over the next 8 minutes determined. B.
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granular accumulation of freely diffusible 5HT occurs under these conditions since such 5HT would be expected to be released on exposure of the platelets to the electric field in the presence of low (lo-9M.) Ca2+. The basal release observed in these experiments is independent of exposure to the field and probably results fran minimal activation of the platelets during This suggestion is supported by the isolation fran platelet-rich plasma. observation (Fig. 3) that this basal release is suppressed by pre-incubation of the platelets with indomethacin. The data obtained using the electric field method provide direct evidence for the importance of a change in cytosolic Ca2+ concentration in the secretory event and hence confirm inferences drawn frcm studies using intact platelets (cf. 6). The dose/response curve describing the Ca2+ dependence of 5HT secretion in the platelet closely approximates that obtained for secretion of nor-adrenaline and dopamine-&hydroxylase fran adrenal medullary cells and for the secretory event involving the cortical granules of sea urchin eggs (11,141. Although such a ccmparison suggests a direct role for Ca2+ in platelet exocytosis the data now available are inadequate to sustain this postulate. The absence of a protein of defined characteristics in the platelet amine storage granule makes it impossible to demonstrate differential release by Ca 2+ of granular versus cytosolic proteins fran the accessed cells. Furthermore we cannot at the manent be certain that in such cells Ca2+ is acting directly on the secretory apparatus since the platelet contains several major Ca2+ stores and considerable precedent exists for a process of Ca2+-induced Ca2+ release in other tissues (15). Thus the dose/response curve observed could represent that for Ca2+ release rather than for the exocytotic event itself. We can however exclude a role for PGG2 or TxA2 since blockade of cyclooxygenase by indanethacin has no effect on the response to Ca2+ (Fig. 3). Despite these limitations it is clear that the electric field technique can be applied to the study of secretion from human platelets. Further studies are in progress to define the role of Ca2+ in secretion from the protein storage granules and the secondary lysosoines, and to examine the participation of this cation in the aggregatory response.
REFERENCES
1.
FEINMAN, R.D. and DETWILER, T.C. Kinetics of the thranbin-induced release of ATP by platelets. Canparison with release of calcium. Biochemistrv, 12, 2462-2468, 1973.
2.
FEINMAN, R.D. and DETWILER, divalent cation ionophores.
3.
ROBBLEE, L.S. and SHEPRO, D. The effect of external calcium and lanthanum on platelet calcium content and on the release reaction. Biochim. Biophys. Acta, 436, 448-459, 1976.
4.
ROBBLEE, L.S., SHEPRO, D., BELAMARICH, calcium flux and the release reaction.
5.
SCRUTTCN, M.C. and EGAN, C.M. Divalent cation requirements for aggregation of human blood platelets and the role of the anti-coagulant. Thranbosis. Res .,14, 713-727, 1979.
T.C. Platelet secretion induced by Nature (Land.), 249, 172-173, 1974.
F.A. and TOWLE, C. Ser. Haematol .,&
Platelet 311-316, 1973.
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6.
MACINTYRE, D.E. The Platelet Release Reaction. In: Platelets in Biology and Pathology. J.L. Gordon (Ed.) Amsterdam: Elsevier/NorthHolland Bianedical Press, 1976, p.61-85.
7.
BAKER, P.F. and KNIGHT, D.E. Calcium-dependent exocytosis in bovine adrenal medullary cells with leaky plasma membranes. Nature (Land.), 276, 620-622, 1978.
8.
ASTER, R.H. and JANDL, J.H. Platelet sequestration in man. J. Clin. Invest., 43, 843-855, 1964.
I.
9.
KNIGHT, D.E. Accessing the interior of cells by exposure to intense electric fields. In: Techniques in the Life Sciences; Techniques in Cellular Physiology. Baker, P.F. (Ed.) Elsevier/North Holland Bianedical Press. Vol. 1, In Press.
Methods.
10.
ZIMMERMANN, U., PILWAT, G. and RIEMANN, F. Dielectric breakdown of cell 881-899, 1974. membranes. &iophvsical J.,&
11.
BAKER, P.F. and KNIGHT, D.E. Calcium-dependence of catecholamine release fran bovine adrenal medullary cells after exposure to intense electric fields. J. Physiology, In Press.
12.
NEUMANN, E. and ROSENHECK, K. Permeability changes induced by electric impulses in vesicular membranes. J. Membrane Biology, 10, 279-290, 1972.
13.
MILLS, D.C.B., ROBB, I.A. and ROBERTS, G.C.K. The release of nucleotides, 5-hydroxytryptamine and enzymes fran human blood platelets during aggregation. J. Physiology, 195, 715-729, 1968.
14.
BAKER, P.F., KNIGHT, D.E. and WHITAKER, M.J. The relation between ionised calcium and cortical granule exocytosis in eggs of the sea urchin Echinus esculentus. Proc. Roy. Sot. B., 207, 149-161, 1980.
15.
FABIATO, A. and FABIATO, F. Calcium-induced release of calcium fran the sarcoplasmic reticulum of skinned cells frau adult human, dog, cat, rabbit, rat and frog hearts and from fetal and new-born rat ventricles. Ann. N.Y. Acad. Sci., 307, 491-522, 1978.
DIRECT EVIDENCE FOR A ROLE FOR CA2+ IN AMINE STORAGE GRANULE SECRETION BY HUMAN PLATELETS
D.E. KNIGHT and M.C. SCRUTION Departments of Physiology and Biochemistry King's College Strand London WC2R 2LS, U.K.
(Received 22.8.1980; in revised form 29.10.1980. Accepted by Editor A.L. Bloom) ABSTRACT Exposure of washed human platelets to a high voltage electric field renders these cells permeable to small molecules such as Ca EGTA. Such accessed platelets secrete 5-hydroxytryptamine (SHT) when challenged with lo-5M Ca2+ but do not release lactate dehydrogenase under these conditions. The secretion of 5HT induced by Ca2+ requires MgATP but is independent of synthesis of prostaglandinendoperoxides. Half maximal secretion of 5HT is observed in the presence of 1.9)rM Ca2+. These data suggest a direct involvement of Ca2+ in platelet amine granule secretion.
INTRODUCTION
Secretion of adenine nucleotides and 5-hydroxytryptamine (5HT) from the amine storage granules of human blood platelets can be induced by a strong agonist such as thrcmbin or by the divalent cation ionophore A-23187 in the absence of aggregation and in media devoid of Ca2+ (l-3). However, when weak agonists such as ADP or adrenaline are employed observation of amine storage granule secretion is dependent on a prior aggregatory response and on the presence of Ca2+ in the medium at concentrations approaching the mM range (4,5). It is therefore inferred fran these and other observations that Ca2+ is directly involved in the pranotion of platelet secretion as is the case for other cells (cf. 6). Direct experimental evidence for such a role is however lacking for the platelet, since studies of the secretory event are limited by the technical difficulties involved in by-passing the transport systems of the plasma membrane while still retaining a preparation which possesses reasonable physiological integrity. We have therefore
Key Words: Platelets, Secretion, Calcium 437
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modified electric field technique which has been employed in studies of the role of Ca2+ in adrenalmedullary secretion (7) for use with the blood platelets, and have been able to obtain both direct evidence for the role of Ca2+ in secretion of 5HT fran human platelets and also an estimate of the range of intracellular Ca2+ concentration over which secretion is induced.
METHODS
Blood obtained fran healthy drug free volunteers was anticoagulated by addition of 1/6th volume of acid citrate-dextrose (8) and platelet-rich plasma prepared by centrifugation for 20 mins at 310 x g and then for 5 mins at 340 x g. After incubation for 30 mins at 37oC with 0.5 uCi [14C] - 5BT (creatinine sulphate) per 20 ml. of platelet-rich plasma platelets were collected by centrifugation for 25 mins at 415 x g and were resuspended in 0.28 M sucrose containing 20 mM K+PIPES, 20 mM K+ glutamate, 5 mM ATP, 7 mM MgCl2 and 5 mM glucose at pH 6.6 (buffer A). In a few experiments the resuspension medium contained 0.16 M K+ glutamate, 20 mM K+ PIPES, 5 mM ATP, 7 mM MgC12 and 5 mM glucose at pH 6.6 (buffer B). After addition of 10 mM EGTA the platelet plasma membrane was rendered leaky by exposure of these cells at 2OoC to an electric field generated by discharge of a 4.5 uF capacitor (previously charged to 1.5 KV) through 1.25 ml of platelet suspension (ca. 108 cells per ml.) contained between two parallel electrodes with a 1 mm gap. These conditions generate an electric field of 15 KV cm-l which decays with a calculated time constant of approximately 4 useconds (buffer B) or 30 useconds (buffer A). In theory exposure of the platelets to such a field should place a maximal potential difference of 2.25 volts across the plasma membrane at two sites, one at each end of the cell (9,lO). Dielectric breakdown, and hence localised membrane disruption, occurs at these sites as a result of this potential difference. The potential difference becomes established within the usecond exposure to the electric field since the time taken to charge up the membrane is calculated to be in the nanosecond range (9). Exposure to further discharges will be likely to produce additional points of membrane breakdown since the orientation of the cell will change with respect to the electric field. For adrenal medullary cells the diameter of the "holes" produced by similar membrane potentials has been estimated as being between 2 and 4 nm (9). The increase in temperature resulting frcm exposure of the platelet suspension to ten 15 KV discharges did not exceed 5oC. The extent of 5BT release was estimated from the [14C] content of the supernatant fraction after removal of the platelets by centrifugation at 8,000 x g. for 2 min. The [14C]content of the supernatant fraction remains constant after ccmpletion of release indicating that re-uptake of 5HT does not occur in platelets which have been exposed to the electric field. The Ca2+ concentration in the Ca EGTA buffers was estimated as described previously (11).
RESULTS
Two experimental approaches demonstrate that exposure of human platelets to an electric field as described above leads to localised, irreversible
439
PLATELET CALCIUM
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c (0
60.
2 I _r
40. 8 x In m
lr-? P
20-
o’ .
0
5
10
15
20
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FIG. 1 Efflux and influx in leaky platelets A. Rb efflux from platelets rendered leaky by a single exposure to an electric field of 4 us duration and intensity 2.5 KVcm-1 (0 1, and 7.5 KVcm-1 (+ 1. Cells not exposed to the field (0 ). The suspending medium was plasma and the temperature 23OC. 45Ca EGTA influx into cells after 10 exposures to an electric B. field of 15 KVQU-1 ( V = 4 ps) ( ?? ). Cells not exposed to electric fields (0 ). Medium: Buffer B containing 10 mM EGTA and trace amaunts of 45Ca and 3H2O. Calculated free calcium a lo-gM. After exposure to the electric field, aliquots (0.2 ml.) of the platelet suspension were layered onto 0.2 ml of silicone oil (Versilube F50) and the cells separated by centrifugation at 8,000 x g for 2 mins.
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i ??-
+-+-
lo-‘M
10S5M
Ca
10S5M
of
Ca
10-QM Ca +_
?? -•
nos.
Ca
discharges
(10 KVCti’)
FIG. 2 Ca2+ dependence of 5BT release The ordinates are expressed as % of the total 5HT in the suspension. Medium: Buffer B containing 10 mM CaEGTA to give calculated free Ca2+ of 10m5M ( 0 , 0 ) or lo-9M (a , 0 1. Open symbols: platelets not exposed to electric fields. A. 5HT in the supernatant fraction at different times after subjecting platelets to 10 exposures of 10 ICVC~-1 ( q = 4 us.) B. 5HT release in the supernatant fraction 5 minutes after subjecting platelets to the indicated number of discharges of 10 KVcm-1 (q = 4 us).
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First, platelets pre-loaded with 86Rb by incubation at membrane breakdown. 23oC for 30 mins rapidly release this radiolabel after a single exposure to a 7.5 KV cm-1 field (Fig. IA). Prolonged incubation following this exposure does not lead to re-uptake of 96Rb indicating that if membrane resealing has occurred, this process does not restore the ability to perform monoExposure to a lower field valent cation uptake using the Na+/K+ ATPase. (2.5 KVcm-1) causes a slower rate of S6Rb efflux from the platelets (Fig. 1A) suggesting a relationship between the strength of the applied field and the Second, platelets which do not normally take extent of membrane breakdown. up 45Ca in the resting state even in the absence of EGTA can be induced to take up 45Ca-EGTA rapidly in a system containing approximately 10sgM Ca2+ (free) on exposure to 10 discharges of a 15 ~vcm-1 field. In a system preequilibrated with 3H2O canparison of the 45Ca and 3H spaces before and after exposure to the field indicates that in excess of 65% of the intracellular environment of the platelets is made accessible to Ca-EGTA by this treatment (Fig. 1B). A slower rate, and possibly a decreased extent, of accessibility to 45Ca-EGTA is observed if the number of discharges or the intensity of the field is decreased. When platelets, previously loaded with [14C] - 5HT as described in "Methods", are rendered accessible to a Ca-EGTA buffer (10 mM EGTA) containing 70% of the [14C] 5HT is released to the 10m5M Ca2+ (free) approximately medium. In contrast, less than 5% release of [14C] - 5HT is observed when the Ca-EGTA buffer employed contains lo-9M Ca2+ (free) (Fig. 2A). This Ca2+-dependent release of [14C] - 5HT is rapid (Fig. 2A) and is not dependent on the discharge regime employed provided that the number of discharges are sufficient to ensure maximal accessibility to the Ca-EGTA buffer (Fig. 2B). The dependence of the release of [l+]-5HT on Ca2+ is confirmed by measurement of a dose/response curve with respect to Ca 2+ (free) concentration over the range 10sg to lo-5M Ca2+, (free) using the 10 mM Ca-EGTA buffer system. Fran the log dose/response relationship (Fig. 3) observed in several such experiments the Ca2+ (free) concentration required to induce 50% maximal release of [14C] - 5HT is obtained as 1.9 + 0.9 uM (S.E.M.) (n = 9). Fig. 3 also demonstrates that preincubation of the platelets with 10 pM indomethacin does not significantly affect the extent of Ca2+ -dependent 5HT release or the PreCa2' (free) concentration at which 50% maximal release is observed. incubation with indomethacin does however depress the extent of [ldc] - SHT release observed in the presence of lo-9M Ca2+ (free) and produces an approximately canparable depression of the extent of [14~] - 5HT release across the entire range of Ca2+ (free)concentration examined. Hence indomethacin is effective in this system. This observation therefore validates the conclusion that Ca2+ dependent 5HT release is unaffected by blockade of cyclooxygenase. TWO observations suggest that Ca 2+-dependent release of [14C] - 5HT fran human platelets is an exocytotic event. First, release of [14c] - 5HT induced by Ca2+ is not accanpanied by release of the cytosolic marker enzyme, lactate dehydrogenase (LDH) (Fig. 4). Second, observation of Ca2+dependent release of [14C] - ~HT is dependent on the availability of MgATP. The requirement for MgATP can be demonstrated by exposing platelets to the electric field in a buffer system containing 10~9M Ca2+ (free) and either no MgATP or 5 mM MgATP. After 5 minutes incubation in these buffers the accessed cells are then challenged by increasing the Ca2+ (free) concentration to 10s5M. Fig. 5A indicates that platelets exposed to the electric field in a buffer containing no MgATP rapidly lose their ability to release
PLATELET CALCIUM
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60-
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-oo-
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Ca2+ CM?
I
-5
FIG. 3 Ca*+
activation curve for 5HT release
A suspension of platelets in medium A containing 0.3 mM EGTA was subjected to 5 discharges of 20 KVcm-1 ( q = 30 us) and aliquots then challenged with a range of 10 mM CaEGTA buffers. The 5HT in the supernatant fraction 5 minutes later was determined and is expressed on the ordinate as % of that in the suspension. Half the platelet suspension was preincubated with 10 PM indanethacin for 5 minutes before being exposed to the electric field ( 0 1. Platelets not incubated with indcmethacin ( 0 ).
- 5HT when the (free) Ca*+ concentration is subsequently increased to r% 10-5M. The extent to which responsiveness is lost is related to the degree of exposure to the electric field and hence possibly to the extent to which endcgeneous MgATP is lost. However loss of responsiveness is not observed if 5 mM MgATP is present in the suspending medium (Fig. 5A)_and after incubation in a medium lacking MgATP responsiveness to a Ca‘+ challenge may be restored by addition of MgATP. Fig. 5B shows a dose/response curve for such a restoration of responsiveness by MgATP for platelets exposed to 5 discharges of a 20 KVC~-~ field in a buffer lacking MgATP. Half maximal restoration of responsiveness is observed in the presence of 0.9 m!!MgATP.
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PLATELET CALCIUM
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30
80
14C-5H T :/: 1:
60
40
50
40
I-
. /
2c )-
. 20
/
-8-
-.
.-----: 0
o-o+ 0
I
LDH / 0
I
6i2+
-7
LOG
-o&Oo I
I
-5
-4
0
(M)
FIG. 4 Ca2+ activation curve for 5HT ( 0
) and LDH ( 0 ) release.
A suspension of platelets in medium A containing 0.3 mM EGTA was subjected to a single discharge of 20 KVcm-1 ( ‘f = 30~s) and aliquots subsequently challenged with a range of 10 mM CaEGTA buffers. 5HT and LDH in the supernatant fraction were determined 5 minutes later and are expressed as percentages of the totals in the suspension.
DISCUSSION The studies described here demonstrate that the electric field technique which has previously been employed to probe secretory mechanisms in the adrenal medullary cells can also successfully be employed with only minor modification to examine this process in human blood platelets which are among the smallest of mammalian cells. Although the applied field is necessarily considerably more intense than that employed for adrenal medullary cells (7) the data obtained indicate clearly that functional integrity is preserved in the platelet at least in respect to the secretory mechanism. Minimal release of 5HT.results from exposure to the electric field in a buffer containing 10WgM Ca2+ indicating that as predicted (9,12) no significant breakdown of the membranes of the amine storage granules results fran exposure to the electric field. Furthermore the maximal extent of 5HT secretion obtained when accessed cells are challenged with 10m5M Ca2+ (free) is similar to that observed on stimulation of intact platelets with a saturating concentration of collagen, adrenaline or thranbin (13). These data also demonstrate that little, if any, extra-
444
PLATELET CALCIUM
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0.2
O-5
1
2
5
Mg-ATP (mM) FIG. 5 MgATP requirement for 5ET release A. Platelets were suspended in (i) Medium A containing 0.4 mM EGTA (+ 1, and in (ii) Medium A containing 0.4 W-lEGTA but without the The two suspensions were subjected to the 5 mM MgATP (0 ). indicated number of discharges at 20 Kvcm-1 ( q = 30 us) and were challenged 5 mins. later with 10 mM CaEGTA buffers corresponding to 10egM Ca2+. After a further 5 minutes the extracellular 5ET was determined, the difference in the levels for the two EGTA challenges representing the Ca2+ dependent release. Cells in Medium A containing 0.4 mM EGTA but no MgATP were -1 (q* 30 us) and one minute subjected to 5 exposures of 20 ICVcm later aliquots were incubated with various concentrations of MgATP. After a further 10 minutes the cells were challenged with 10 mM CaEGTA corresponding to 10-g and 10a5M Ca2+, and the Ca2+ dependent release over the next 8 minutes determined. B.
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PLATELET CALCIUM
granular accumulation of freely diffusible 5HT occurs under these conditions since such 5HT would be expected to be released on exposure of the platelets to the electric field in the presence of low (lo-9M.) Ca2+. The basal release observed in these experiments is independent of exposure to the field and probably results fran minimal activation of the platelets during This suggestion is supported by the isolation fran platelet-rich plasma. observation (Fig. 3) that this basal release is suppressed by pre-incubation of the platelets with indomethacin. The data obtained using the electric field method provide direct evidence for the importance of a change in cytosolic Ca2+ concentration in the secretory event and hence confirm inferences drawn frcm studies using intact platelets (cf. 6). The dose/response curve describing the Ca2+ dependence of 5HT secretion in the platelet closely approximates that obtained for secretion of nor-adrenaline and dopamine-&hydroxylase fran adrenal medullary cells and for the secretory event involving the cortical granules of sea urchin eggs (11,141. Although such a ccmparison suggests a direct role for Ca2+ in platelet exocytosis the data now available are inadequate to sustain this postulate. The absence of a protein of defined characteristics in the platelet amine storage granule makes it impossible to demonstrate differential release by Ca 2+ of granular versus cytosolic proteins fran the accessed cells. Furthermore we cannot at the manent be certain that in such cells Ca2+ is acting directly on the secretory apparatus since the platelet contains several major Ca2+ stores and considerable precedent exists for a process of Ca2+-induced Ca2+ release in other tissues (15). Thus the dose/response curve observed could represent that for Ca2+ release rather than for the exocytotic event itself. We can however exclude a role for PGG2 or TxA2 since blockade of cyclooxygenase by indanethacin has no effect on the response to Ca2+ (Fig. 3). Despite these limitations it is clear that the electric field technique can be applied to the study of secretion from human platelets. Further studies are in progress to define the role of Ca2+ in secretion from the protein storage granules and the secondary lysosoines, and to examine the participation of this cation in the aggregatory response.
REFERENCES
1.
FEINMAN, R.D. and DETWILER, T.C. Kinetics of the thranbin-induced release of ATP by platelets. Canparison with release of calcium. Biochemistrv, 12, 2462-2468, 1973.
2.
FEINMAN, R.D. and DETWILER, divalent cation ionophores.
3.
ROBBLEE, L.S. and SHEPRO, D. The effect of external calcium and lanthanum on platelet calcium content and on the release reaction. Biochim. Biophys. Acta, 436, 448-459, 1976.
4.
ROBBLEE, L.S., SHEPRO, D., BELAMARICH, calcium flux and the release reaction.
5.
SCRUTTCN, M.C. and EGAN, C.M. Divalent cation requirements for aggregation of human blood platelets and the role of the anti-coagulant. Thranbosis. Res .,14, 713-727, 1979.
T.C. Platelet secretion induced by Nature (Land.), 249, 172-173, 1974.
F.A. and TOWLE, C. Ser. Haematol .,&
Platelet 311-316, 1973.
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PLATELET CALCIUM
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6.
MACINTYRE, D.E. The Platelet Release Reaction. In: Platelets in Biology and Pathology. J.L. Gordon (Ed.) Amsterdam: Elsevier/NorthHolland Bianedical Press, 1976, p.61-85.
7.
BAKER, P.F. and KNIGHT, D.E. Calcium-dependent exocytosis in bovine adrenal medullary cells with leaky plasma membranes. Nature (Land.), 276, 620-622, 1978.
8.
ASTER, R.H. and JANDL, J.H. Platelet sequestration in man. J. Clin. Invest., 43, 843-855, 1964.
I.
9.
KNIGHT, D.E. Accessing the interior of cells by exposure to intense electric fields. In: Techniques in the Life Sciences; Techniques in Cellular Physiology. Baker, P.F. (Ed.) Elsevier/North Holland Bianedical Press. Vol. 1, In Press.
Methods.
10.
ZIMMERMANN, U., PILWAT, G. and RIEMANN, F. Dielectric breakdown of cell 881-899, 1974. membranes. &iophvsical J.,&
11.
BAKER, P.F. and KNIGHT, D.E. Calcium-dependence of catecholamine release fran bovine adrenal medullary cells after exposure to intense electric fields. J. Physiology, In Press.
12.
NEUMANN, E. and ROSENHECK, K. Permeability changes induced by electric impulses in vesicular membranes. J. Membrane Biology, 10, 279-290, 1972.
13.
MILLS, D.C.B., ROBB, I.A. and ROBERTS, G.C.K. The release of nucleotides, 5-hydroxytryptamine and enzymes fran human blood platelets during aggregation. J. Physiology, 195, 715-729, 1968.
14.
BAKER, P.F., KNIGHT, D.E. and WHITAKER, M.J. The relation between ionised calcium and cortical granule exocytosis in eggs of the sea urchin Echinus esculentus. Proc. Roy. Sot. B., 207, 149-161, 1980.
15.
FABIATO, A. and FABIATO, F. Calcium-induced release of calcium fran the sarcoplasmic reticulum of skinned cells frau adult human, dog, cat, rabbit, rat and frog hearts and from fetal and new-born rat ventricles. Ann. N.Y. Acad. Sci., 307, 491-522, 1978.